原生质体紫外诱变选育高产抗菌脂肽菌株及其活性物质的研究

本研究以实验室自主分离的枯草芽孢杆菌mutHS-301为出发菌株,通过原生质体紫外诱变选育出高产抗菌脂肽突变菌株,并对其产生的抗菌脂肽提取物进行单组分分离纯化及对黄曲霉抑制作用进行初步研究。结果表明,在溶菌酶浓度为0.5 mg/mL,酶解时间为15 min,酶解温度为37℃条件下,获得原生质体的形成率和再生率效果最佳。采用紫外照射时间60 s进行该原生质体诱变,经筛选获得一株遗传性状稳定的高产抗菌脂肽菌株,命名为mutHS-539。研究表明,该突变株mutHS-539发酵上清液对副溶血性弧菌和金黄色葡萄球菌抑菌直径较原始菌mutHS-301分别提高了21.49%和21.05%,提取得到的抗菌脂肽产量较原始菌提高了40%。利用制备型硅胶板对发酵提取物进行分离纯化得到四种组分,分别为a、b、c和d;进一步检测对黄曲霉的抑菌活性,结果发现只有组分d对黄曲霉具有显著的抑制作用。经RP-HPLC分析及液质联用数据比对,该组分d的主要成分为杆菌霉素D。该抗菌脂肽提取物对黄曲霉抑制作用的研究显示,当抗菌脂肽浓度为0.2 mg/mL时能有效抑制黄曲霉菌丝的生长,抑制率达到了74.22%,且对黄曲霉孢子的致死浓度为0.8 mg/mL。

Screening of high-yielding antibacterial lipopeptides by protoplast UV mutagenesis and its inhibitory effects on Aspergillus flavus

In this project,Bacillus subtilis mutHS-301 isolated from the laboratory was used as the initial strain.Firstly,the high-yielding antibacterial lipopeptide strain was screened by protoplast UV mutagenesis method.The formation rate and regeneration rate of protoplasts were optimized under the conditions of 0.5 mg/mL of lysozyme concentration,15 min of enzymatic hydrolysis time and 37℃of hydrolysis temperature.Then the protoplast mutagenesis was carried out by UV irradiation for 60 s.As a result of screening,a mutant strain named mutHS-539 was obtained with the characteristics of genetically stable antibacterial lipopeptides.Further study found that the inhibitory effects of the strain mutHS-539 against Vibrio parahaemolyticus and Staphylococcus aureus were increased by 21.49%and 21.05%,respectively,compared with those of the original strain mutHS-301.The antibacterial lipopeptide yield of the strain mutHS-539 was increased by 40%compared with that of the original strain.Secondly,the fermentation supernatants of the strain mutHS-539 were extracted,and the crude extract was separated and purified.The four components were separated and purified by preparative silica gel plates,which were named a,b,c and d,respectively.The antibacterial activity test showed that only component d had significant inhibitory effect on Aspergillus flavus.Based on analysis of RP-HPLC and data comparison of the liquid chromatography-mass spectrometry,the main substance of the component d was Bacillusmycin D.Finally,the active extract produced by the fermentation of the strain mutHS-539 was further applied to the antifungal test.The results revealed that the antibacterial lipopeptide concentration of 0.2 mg/mL could effectively inhibit the growth of Aspergillus flavus with the inhibition rate of 74.22%,and the lethal concentration to Aspergillus flavus spores was 0.8 mg/mL.